The RNA pseudoknots in foot and mouth disease virus are dispensable for genome replication but essential for the production of infectious virus

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5 ' untranslated region (5 ' UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions. Author summaryFoot-and-mouth disease (FMD) is a highly contagious viral disease posing a constant threat to the global livestock industry, as it remains endemic in many parts of the world. The development of novel control measures for FMD could have major economic and social value worldwide, however, this will require a deeper understanding of the molecular mechanisms of FMDV replication. A number of features of the viral RNA genome remain poorly understood, including the role of a series of putative pseudoknot (PK) structures, where the RNA is predicted to fold back on itself. In this study, we confirmed the conservation and structure of these PKs in the FMDV genome and showed that the presence of at least one is essential for production of infectious virus particles. However, the presence of two or more PKs was advantageous in replication competition experiments. These results correlate with the observation that genomes of all known FMDV field isolates include at least two PK structures. We suggest roles for the PKs in both replication and virion assembly.